CN102659549B - Method for extracting brevifolin from Relinqing granula raw material and method for preparation quality control - Google Patents
Method for extracting brevifolin from Relinqing granula raw material and method for preparation quality control Download PDFInfo
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- FBUBVLUPUDBFME-UHFFFAOYSA-N Xanthoxylin Chemical compound COC1=CC(O)=C(C(C)=O)C(OC)=C1 FBUBVLUPUDBFME-UHFFFAOYSA-N 0.000 title claims abstract description 60
- 238000000034 method Methods 0.000 title claims abstract description 59
- FXVRSKPFWVYCPN-UHFFFAOYSA-N brevifolin Natural products OC1=C(O)C(O)=CC(C(O2)=O)=C1C1=C2C(=O)CC1 FXVRSKPFWVYCPN-UHFFFAOYSA-N 0.000 title claims abstract description 30
- 239000002994 raw material Substances 0.000 title claims abstract description 25
- 238000002360 preparation method Methods 0.000 title abstract description 17
- 238000003908 quality control method Methods 0.000 title abstract description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 90
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 48
- 239000002904 solvent Substances 0.000 claims abstract description 26
- 238000000926 separation method Methods 0.000 claims abstract description 25
- 239000000203 mixture Substances 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 14
- OXGUCUVFOIWWQJ-HQBVPOQASA-N quercitrin Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1OC1=C(C=2C=C(O)C(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O OXGUCUVFOIWWQJ-HQBVPOQASA-N 0.000 claims abstract description 12
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims abstract description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 17
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 15
- 239000002245 particle Substances 0.000 claims description 15
- 239000006286 aqueous extract Substances 0.000 claims description 13
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 12
- 238000001514 detection method Methods 0.000 claims description 10
- 230000005526 G1 to G0 transition Effects 0.000 claims description 9
- 239000000706 filtrate Substances 0.000 claims description 9
- 239000002024 ethyl acetate extract Substances 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 6
- 238000007654 immersion Methods 0.000 claims description 5
- 238000001704 evaporation Methods 0.000 claims description 4
- 230000008020 evaporation Effects 0.000 claims description 4
- 238000004090 dissolution Methods 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 3
- 238000013517 stratification Methods 0.000 claims description 3
- 238000007670 refining Methods 0.000 claims description 2
- 239000000284 extract Substances 0.000 abstract description 11
- 238000012544 monitoring process Methods 0.000 abstract description 4
- 241000764065 Persicaria capitata Species 0.000 abstract description 3
- LUJAXSNNYBCFEE-UHFFFAOYSA-N Quercetin 3,7-dimethyl ether Natural products C=1C(OC)=CC(O)=C(C(C=2OC)=O)C=1OC=2C1=CC=C(O)C(O)=C1 LUJAXSNNYBCFEE-UHFFFAOYSA-N 0.000 abstract description 3
- PUTDIROJWHRSJW-UHFFFAOYSA-N Quercitrin Natural products CC1OC(Oc2cc(cc(O)c2O)C3=CC(=O)c4c(O)cc(O)cc4O3)C(O)C(O)C1O PUTDIROJWHRSJW-UHFFFAOYSA-N 0.000 abstract description 3
- OXGUCUVFOIWWQJ-XIMSSLRFSA-N acanthophorin B Natural products O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@H]1OC1=C(C=2C=C(O)C(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O OXGUCUVFOIWWQJ-XIMSSLRFSA-N 0.000 abstract description 3
- 238000000605 extraction Methods 0.000 abstract description 3
- 239000008187 granular material Substances 0.000 abstract description 3
- OEKUVLQNKPXSOY-UHFFFAOYSA-N quercetin 3-O-beta-D-glucopyranosyl(1->3)-alpha-L-rhamnopyranosyl(1->6)-beta-d-galactopyranoside Natural products OC1C(O)C(C(O)C)OC1OC1=C(C=2C=C(O)C(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O OEKUVLQNKPXSOY-UHFFFAOYSA-N 0.000 abstract description 3
- QPHXPNUXTNHJOF-UHFFFAOYSA-N quercetin-7-O-beta-L-rhamnopyranoside Natural products OC1C(O)C(O)C(C)OC1OC1=CC(O)=C2C(=O)C(O)=C(C=3C=C(O)C(O)=CC=3)OC2=C1 QPHXPNUXTNHJOF-UHFFFAOYSA-N 0.000 abstract description 3
- 239000013558 reference substance Substances 0.000 abstract 1
- 239000000463 material Substances 0.000 description 24
- 239000012071 phase Substances 0.000 description 22
- 238000004128 high performance liquid chromatography Methods 0.000 description 15
- 239000000243 solution Substances 0.000 description 15
- 238000012360 testing method Methods 0.000 description 15
- 239000003814 drug Substances 0.000 description 14
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 12
- 239000000843 powder Substances 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 229940079593 drug Drugs 0.000 description 8
- 238000011160 research Methods 0.000 description 8
- 239000000126 substance Substances 0.000 description 7
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- 238000003809 water extraction Methods 0.000 description 6
- 238000005481 NMR spectroscopy Methods 0.000 description 5
- 230000002411 adverse Effects 0.000 description 5
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- 239000000178 monomer Substances 0.000 description 5
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- 235000011114 ammonium hydroxide Nutrition 0.000 description 4
- 238000010262 high-speed countercurrent chromatography Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 239000000470 constituent Substances 0.000 description 3
- 239000012074 organic phase Substances 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 3
- 239000010452 phosphate Substances 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- 241001251200 Agelas Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 208000004880 Polyuria Diseases 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
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- 230000035619 diuresis Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 description 2
- 241000411851 herbal medicine Species 0.000 description 2
- 238000005192 partition Methods 0.000 description 2
- 238000011155 quantitative monitoring Methods 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- 238000005507 spraying Methods 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 101000623895 Bos taurus Mucin-15 Proteins 0.000 description 1
- 241000463291 Elga Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241001529246 Platymiscium Species 0.000 description 1
- 241000219050 Polygonaceae Species 0.000 description 1
- 241000205407 Polygonum Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000003260 anti-sepsis Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000002451 electron ionisation mass spectrometry Methods 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
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- 235000004515 gallic acid Nutrition 0.000 description 1
- 229940074391 gallic acid Drugs 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
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- 238000004811 liquid chromatography Methods 0.000 description 1
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- 239000007791 liquid phase Substances 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
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- 238000010606 normalization Methods 0.000 description 1
- 239000011236 particulate material Substances 0.000 description 1
- 150000007965 phenolic acids Chemical class 0.000 description 1
- 235000009048 phenolic acids Nutrition 0.000 description 1
- 239000004810 polytetrafluoroethylene Substances 0.000 description 1
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000013094 purity test Methods 0.000 description 1
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- 238000002137 ultrasound extraction Methods 0.000 description 1
- 230000002485 urinary effect Effects 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
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Abstract
The invention relates to a method for extracting brevifolin and/or quercitrin from a Relinqing granula raw material such as polygonum capitatum water extract and also relates to a method for quality control of Relinqing granules by brevifolin. The method for extracting brevifolin and/or quercitrin utilizes a high-speed countercurrent chromatograph (HSCCC) and a mixture as a HSCCC solvent system to realize separation, wherein the mixture comprises n-hexane, ethyl acetate, methanol and water and a ratio of n-hexane, ethyl acetate, methanol to water is 1: (4-6): (0.8-1.2): (4-6). The method for extracting brevifolin realizes high purity and high yield extraction of brevifolin from a Relinqing granula raw material and brevifolin can be used as a reference substance in quality monitoring of a polygonum capitatum preparation such as Relinqing granules.
Description
Technical field
The invention belongs to technical field of traditional Chinese medicines, relate to from heat and drench the method for extracting brevifolin the raw material of clear particle, for example from the Herba Polygoni Capitati aqueous extract, extract the method for brevifolin, also relate to from heat and drench the method for extracting brevifolin and Quercitroside the raw material of clear particle or Herba Polygoni Capitati aqueous extract.The present invention also further relates to the use brevifolin heat is drenched to the method that clear granular preparation carries out quality monitoring.
Background technology
Dry herb or over-ground part that Herba Polygoni Capitati is polygonaceae plant Herba Polygoni Capitati Polygonum capitatum Buch.-Ham.ex D.Don, record in " the national quality of medicinal material standard of Guizhou Province's Chinese medicinal materials " version in 2003, the effects such as tool clearing heat and promoting diuresis, antisepsis and anti-inflammation, inducing diuresis for treating stranguria syndrome (Guizhou Province food and medicine Surveillance Authority. the national quality of medicinal material standard [M] of Guizhou Province's Chinese medicinal materials. Kweiyang: Guizhou science and technology press, 2003:147).Herba Polygoni Capitati is Guizhou seedling medicine commonly used, is one of the kind of " the seven large Chinese medicine industrial chains " of Guizhou Province's modernization of Chinese medicine " the six seedlings medicines " given priority to and emphasis cultivation and development.
The heat that the Herba Polygoni Capitati of take is main raw material is drenched clear particle and is used for the treatment of clinically urinary system infection, by " Chinese pharmacopoeia one one of version in 2010 is recorded, but less to the research report of Herba Polygoni Capitati so far both at home and abroad, existing document is tentatively thought the effective constituent that flavonoid, phenolic acids are Herba Polygoni Capitati
[1-4]." Chinese pharmacopoeia version in 2010 is only usingd gallic acid and is drenched the quantitative monitoring index of clear particle as heat, still expects to have more effective reliable evaluation index at present.
The bulk drug (being the Herba Polygoni Capitati aqueous extract, is Powdered thing) that the inventor is devoted to heat is drenched clear particle carries out the full composition analysis of system, expects to find to can be used for the quantitative monitoring 'Relinqing ' novel method that for example heat is drenched clear granular preparation.As everyone knows, the material of monitoring complicated component for example Chinese medical extract need to have clear and definite monitor control index, for example need to have in this Chinese medicine, contain, can obtain, facile reference material, and for a certain Chinese medical extract, if can monitor its quality with the number of chemical material simultaneously, it is more reasonable for the quality of medicine, will to control control than one-component.
Therefore, from heat, drench in the bulk drug (being the Herba Polygoni Capitati aqueous extract, is Powdered thing) of clear particle and find that new chemical substance is still that those skilled in the art expect, in order to control the new approach that provides for drug quality.
Summary of the invention
The object of the invention is to drench as heat the new way that clear particle provides drug quality to control for the Herba Polygoni Capitati formulation example.The inventor finds, the bulk drug that adopts unique method to process the clear granular preparation of heat pouring is the Herba Polygoni Capitati aqueous extract, searching out a kind of novel substance of not reported in medicinal material of polygonum capilalum is brevifolin (brevifolin), and the method has advantages of useful industrial value at the yield aspect this novel substance of acquisition and other chemical monomer.The present invention is based on this discovery and be accomplished.
For this reason, first aspect present invention provides from heat drenches the method for for example, extracting brevifolin (and/or Quercitroside) the raw material (Herba Polygoni Capitati aqueous extract) of clear particle, and the method comprises the following steps:
(1) get the Herba Polygoni Capitati aqueous extract, by 60～95% alcohol immersion 5～24 hours, supersound process 10～120min, filter the filtrate evaporation drying, the resistates water dissolution, filter, filtrate is extracted with ethyl acetate to upper strata colourless, and ethyl acetate layer is merged, evaporation drying, obtain ethyl acetate extract;
(2) step (1) gained ethyl acetate extract is loaded in high-speed counter-current chromatograph (HSCCC), use normal hexane: ethyl acetate: methyl alcohol: the mixture of water (1: 4～6: 0.8～1.2: 4～6) is separated as the HSCCC solvent systems, collects the flow point of 40～90min; Repeat in case of necessity the operation of this separation and collection; Described flow point is steamed and desolventizes, obtain Herba Polygoni Capitati secondary separation raw material thing;
(3) in separating funnel, in ethyl acetate: the dipotassium hydrogen phosphate solution of propyl carbinol: 0.008～0.012mol/L=3.36～5.04: 0.64～0.96: 5 ratio (volume ratio) configures the two-phase solvent system of about 1500mL, stratification Cheng Shangxiang (stationary phase) and lower phase (moving phase), ultrasonic degas;
(4) adopt the type of elution of unicoil, head-to-tail, at first make pipeline be full of stationary phase (upper phase), the main frame forward, then pump into moving phase, treat that moving phase flows out, and when two-phase solvent reaches equilibrium state in post, gets step (2) gained Herba Polygoni Capitati secondary separation raw material thing, be dissolved in isopyknic up and down mutually in, use syringe sampling;
(5) divide and detected with the UV-detector convection current, the detection wavelength is 250～290nm, records color atlas simultaneously, according to color atlas, with fraction collector, automatically collects flow point, and every 3min collects a pipe; Disengaging time is more than 270min;
(6) collect between 30～40min go out peak flow point as flow point I, go out the flow point at peak between 200～260min as flow point II;
(7) remove respectively the solvent of flow point I and flow point II, flow point I and flow point II correspondingly obtain respectively component I and component I I, and optionally further refining to component I and component I respectively, component I is brevifolin, and component I I is Quercitroside.
According to the method for first aspect present invention, wherein, in step (1), ethanol used is 70～90% ethanol, preferably 80% ethanol.
According to the method for first aspect present invention, wherein, in step (1), the time of alcohol immersion is 8-15 hour, for example 10-12 hour.
According to the method for first aspect present invention, wherein, in step (1), the time of supersound process is 15-60min, preferably 20-45min, for example about 30min.
Method according to first aspect present invention, wherein in step (2), described HSCCC solvent systems is normal hexane: ethyl acetate: methyl alcohol: the mixture of water (1: 4.5～5.5: 0.9～1.1: 4.5～5.5), in one embodiment, described HSCCC solvent systems is normal hexane: ethyl acetate: methyl alcohol: water (1: 5: 1: mixture 5).
According to the method for first aspect present invention, wherein, in step (2), collect the flow point of 45～80min, preferably collect the flow point of 53～74min.
According to the method for first aspect present invention, wherein, in step (2), the operation of described separation and collection repeats 2～6 times, preferably repeats 3～5 times, preferably repeats 4 times; And merge collected flow point.
According to the method for first aspect present invention, wherein, in step (3), the ratio of described two-phase solvent system is 3.78～4.62: 0.72～0.88: 5, and the ratio of preferred described two-phase solvent system is 4.2: 0.8: 5.
According to the method for first aspect present invention, wherein, in step (3), the concentration of described dipotassium hydrogen phosphate solution is 0.009～0.011mol/L, is preferably 0.01mol/L.
According to the method for first aspect present invention, wherein, in step (4), the rotating speed of main frame forward is 700～1000rpm, preferably 800～900rpm, preferably 860rpm.
According to the method for first aspect present invention, wherein, in step (4), the flow velocity that pumps into moving phase to pipeline is 1～3mL/min, preferred 1.5～2.5mL/min, preferred about 2mL/min.
According to the method for first aspect present invention, wherein, in step (5), described detection wavelength is 260～280nm, preferably 270～275nm, preferably 272nm.
Second aspect present invention provides the method for a kind of monitoring Herba Polygoni Capitati preparation (for example heat is drenched clear granular preparation) quality, the method comprises the brevifolin product in contrast that use, use high performance liquid chromatography to be detected, measure difference between the measured value of the variation of brevifolin content in the content of brevifolin in said preparation or different batches preparation or every batch of product and average.
According to the method for second aspect present invention, the chromatographic condition of wherein said high performance liquid chromatography is: use C
18chromatographic column, the phosphate aqueous solution that moving phase is methyl alcohol and 0.2%, gradient elution, wherein the gradient ratio of organic phase (methyl alcohol) for example under: 5% (0min)～30% (5min)～65% (20min)～65% (24min)～5% (25min)～5% (30min), the detection wavelength is 272nm.
According to the method for second aspect present invention, the chromatographic condition of wherein said high performance liquid chromatography is: the use stationary phase is Venusil MP C
18chromatographic column (4.6mm * 250mm, 5 μ m, Agela Technologie company), the phosphate aqueous solution that moving phase is methyl alcohol and 0.2%, gradient elution, wherein the gradient ratio of organic phase for example under: 5% (0min)～30% (5min)～65% (20min)～65% (24min)～5% (25min)～5% (30min); The detection wavelength is 272nm, and flow velocity is 1.0mL/min, and column temperature is room temperature, and sample size is 20 μ L.
Below with characteristics, be further described to various aspects of the present invention.All documents that the present invention quotes from, their full content is incorporated to this paper by reference, and if, when the expressed implication of these documents and the present invention are inconsistent, be as the criterion with statement of the present invention.In addition, various terms and phrase that the present invention uses have the general sense of well known to a person skilled in the art, nonetheless, the present invention still wishes at this, these terms and phrase to be described in more detail and to explain, the term of mentioning and phrase, if any inconsistent with known implication, are as the criterion with the implication that the present invention was explained.
In the present invention, the raw material that heat is drenched clear particle can refer to any method and be extracted and obtained extract by the Chinese medicinal materials Herba Polygoni Capitati, and this extract can be used for preparing heat and drenches clear granule.In one embodiment, the raw material of the clear particle of heat pouring refers to the Herba Polygoni Capitati aqueous extract.In one embodiment, the Herba Polygoni Capitati aqueous extract is obtained by following method for making: get Herba Polygoni Capitati boiling secondary, each 1.5 hours, decocting liquid filtered, and filtrate merges, and was concentrated in right amount, filtered, and spraying drying, obtain.
The object of the invention is to explore heat and drench the novel method that clear granular mass is controlled.The inventor is drenched the raw material Herba Polygoni Capitati aqueous extract powder of clear particle from heat, extracts, separates and identify a kind of new constituent-brevifolin (brevifolin), thereby the quality control of for heat, drenching clear particle provides new departure.In a useful method of the present invention, application high-speed countercurrent chromatography (high-speed counter-current chromatography, HSCCC) (solvent systems is normal hexane: ethyl acetate: methyl alcohol: water=1: 5: 1: 5) ethyl acetate extract of Herba Polygoni Capitati water extraction powder to be carried out to the first step separation, flow point to gained carries out secondary separation, (solvent systems is ethyl acetate: the dipotassium hydrogen phosphate solution of propyl carbinol: 0.01mol/L 4.2: 0.8: 5), upper is stationary phase mutually, engine speed is 860rpm, flow velocity is 2.0mL/min, the detection wavelength is 272nm, sample size is 0.25g.Certain embodiments of the invention can obtain two kinds of products that purity is 96.2% and 98.4%, through Structural Identification, are respectively brevifolin and Quercitroside (quercitrin).Wherein brevifolin for separating and obtain first from this platymiscium.The present invention extracts and is separated to brevifolin from Herba Polygoni Capitati water extraction powder; The high-speed countercurrent chromatography used is easy, quick, is suitable for the research of Effective Component of Chinese Medicine and the extraction preparation of monomer.
The accompanying drawing explanation
Fig. 1 has described the color atlas that Herba Polygoni Capitati secondary separation raw material thing, HSCCC gained flow point I and flow point II carry out the HPLC detection, and three kinds of material gained collection of illustrative plates are respectively A, B, the C in figure.
Fig. 2 has described the high speed adverse current chromatogram figure of Herba Polygoni Capitati secondary separation raw material thing
Embodiment
By the following examples, can conduct further description the present invention, yet scope of the present invention is not limited to following embodiment.One of skill in the art can understand, and under the prerequisite that does not deviate from the spirit and scope of the present invention, can carry out various variations and modification to the present invention.The present invention carries out generality and/or concrete description to the material and the test method that use in test.Although, for to realize that many materials and working method that the object of the invention is used are well known in the art, the present invention still does to describe in detail as far as possible at this.
some instruments that use in A, test are known material
Mk5QuilkPrep500 high-speed counter-current chromatograph (Britain AECS company), is furnished with polyfluortetraethylene pipe (PTFE) from post (pipe diameter 2.16mm, separated volume 120mL, the β value is 0.5～0.8), SeriesII constant flow pump (Scientific System company), SPD-10Avp UV-vis detector (Suzhou Shimadzu instrument company), N2000 Data Processing in Chromatography Workstation (Zhejiang University's intelligence reaches Information Technology Co. Ltd).
The Waters1525 high performance liquid chromatograph, Waters717 type automatic sampler, 2996 type diode-array detectors, Empower chromatographic working station (U.S. waters company).
TSQ type triple quadrupole bar Liquid Chromatography-tandem Mass instrument (U.S. San Jose company), Finnigan Surveyor MS pump, Finnigan Surveyor automatic sampler, Xcalibur1.3 workstation, LC Quan data processing software (U.S. Thermo Finnigan company).
Electric constant-temp water-bath (Shantou City, Guangdong Province medical equipment factory), SY-1000E multipurpose thermostatic ultrasonic extractor (Beijing Hongxianglong Biotechnology Development Co., Ltd), DZF-6051SH type vacuum drying oven (beneficial permanent laboratory apparatus company limited), AL204 type electronic balance (Switzerland Metter Toledo company), Dragon-med liquid-transfering gun (Toppette company), RE-3000 Rotary Evaporators (Shanghai Yarong Biochemical Instrument Plant), Anke TGL-16B whizzer (Hai'an booth scientific instrument factory).
TENSOR37 infrared spectrometer (German Bruker company), Bruker AvanceIII400MHz nuclear magnetic resonance analyser (German Bruker company), LCMS-IT-TOF high-resolution mass spectrometer (Japanese Shimadzu company).
Methyl alcohol (chromatographically pure, TEDIA company), ethanol (analytical pure, Tianjin Fu Yu Fine Chemical Co., Ltd), ethyl acetate (analytical pure, Tianjin Fu Yu Fine Chemical Co., Ltd), propyl carbinol (analytical pure, Tianjin recovery development in science and technology company limited), acetic acid (analytical pure, Guangzhou Chemical Reagent Factory), phosphoric acid (analytical pure, Guangzhou Chemical Reagent Factory), ultrapure water (PureLab Ultra ultrapure water system, Britain ELGA company).
The Herba Polygoni Capitati aqueous extract is provided by GuiZhou WeiMen Pharmacy Co., Ltd, and be to obtain with reference to the method for making in the 992nd page of " heat is drenched clear particle " item of version Chinese Pharmacopoeia in 2010, that is: get Herba Polygoni Capitati boiling secondary, each 1.5 hours, decocting liquid filtered, filtrate merges, be concentrated in right amount, filter spraying drying, obtain yield 10.07%.
embodiment 1: the preparation of Herba Polygoni Capitati water extraction powder ethyl acetate extract
Get Herba Polygoni Capitati water extraction powder 150g, spend the night by 80% alcohol immersion of 10 times of amounts, ultrasonic extraction 30min, filter, and filtrate is used to the Rotary Evaporators evaporate to dryness, adds the 150mL water dissolution, filters, and filtrate is extracted with ethyl acetate to upper strata colourless.Ethyl acetate layer is merged, use the Rotary Evaporators evaporate to dryness, obtain Herba Polygoni Capitati water extraction powder ethyl acetate extract (medicinal extract) 12.7g, be sealed in moisture eliminator standby.
embodiment 2: the preparation of Herba Polygoni Capitati secondary separation raw material thing
Take embodiment 1 gained ethyl acetate extract 1.0g, utilize HSCCC solvent systems normal hexane: ethyl acetate: methyl alcohol: water (1: 5: 1: 5) separated, collect the flow point of 53min to 74min, repeat this operation four times, collected flow point is merged, use the Rotary Evaporators solvent evaporated, obtain Herba Polygoni Capitati secondary separation raw material thing 0.25g.
embodiment 3: the purity of high effective liquid chromatography for measuring trial target
high-efficient liquid phase chromatogram condition:stationary phase is Venusil MP C
18chromatographic column (4.6mm * 250mm, 5 μ m, Agela Technologie company), the phosphate aqueous solution that moving phase is methyl alcohol and 0.2%, gradient elution, wherein the gradient ratio of organic phase for example under: 5% (0min)～30% (5min)～65% (20min)～65% (24min)～5% (25min)～5% (30min).The detection wavelength is 272nm, and flow velocity is 1.0mL/min, and column temperature is room temperature, and sample size is 20 μ L.
preparation with the Herba Polygoni Capitati secondary separation raw material thing test liquid of doing the HPLC purity testing:get embodiment 2 gained Herba Polygoni Capitati water secondary separation raw material thing 0.0150g, add 50% methanol-water constant volume in the 10mL volumetric flask, be made into the solution that concentration is 1.50mg/mL, ultrasonic 30min, through the millipore filtration filtration of 0.45 μ m, filtrate for later use.
To above-mentioned Herba Polygoni Capitati secondary separation raw material thing test liquid, and each flow point of HSCCC gained detected, and wherein the purity of product is calculated by area normalization method, and the typical HPLC figure of analysis is shown in Fig. 1.That the figure A in Fig. 1 shows is the HPLC figure of Herba Polygoni Capitati secondary separation raw material thing with above-mentioned HPLC method test, shows I and two chromatographic peaks of II in figure, and it corresponds respectively to component I and component I I.That the figure B in Fig. 1 shows is the HPLC figure that the flow point I in HSCCC tests with above-mentioned HPLC method, has basically only shown the peak of component I in figure.That the figure C in Fig. 1 shows is the HPLC figure that the flow point II in HSCCC tests with above-mentioned HPLC method, has basically only shown the peak of component I I in figure.
embodiment 4: the selection of high speed adverse current chromatogram separation condition
The Golden-rule of selecting according to the high speed adverse current chromatogram solvent systems and the character of material to be separated, this study tour a plurality of solvent systemss, according to partition ratio formula K=CU/CL (CU be upper mutually in the concentration of material to be separated, CL be lower mutually in the concentration of material to be separated) calculate its partition ratio K, the results are shown in Table 1.
Table 1
From table 1 data, in ethyl acetate: propyl carbinol: 0.01mol/L dipotassium hydrogen phosphate solution, ethyl acetate: propyl carbinol: ammoniacal liquor and ethyl acetate: propyl carbinol: in 0.025mol/L sodium hydrogen carbonate solution system, the K value of composition I, II is relatively desirable with corresponding α value.But find ethyl acetate in substantial sepn: propyl carbinol: ammoniacal liquor and ethyl acetate: propyl carbinol: the pH value of 0.025mol/L sodium hydrogen carbonate solution system is unstable, causes the appearance time of material to be separated greatly to postpone, and serious conditions of streaking is arranged.Ethyl acetate is only arranged: propyl carbinol: 0.01mol/L dipotassium hydrogen phosphate solution system appearance time is stable.Test-results also shows, when ethyl acetate: propyl carbinol: during 0.01mol/L dipotassium hydrogen phosphate solution=4.2: 0.8: 5, composition I, II all can obtain good separating effect, and disengaging time is shorter, product purity high (all being greater than 95%), this solvent systems can be used for being separated.
embodiment 5: high speed adverse current chromatogram separates preparation
Configuration two-phase solvent system in separating funnel, by ethyl acetate: propyl carbinol: the proportional arrangement of 0.01mol/L dipotassium hydrogen phosphate solution=4.2: 0.8: 5, the about 1500mL of solution.Stratification, wherein going up is stationary phase mutually, lower is moving phase mutually, ultrasonic degas.Adopt the type of elution of unicoil, head-to-tail, at first make pipeline be full of stationary phase (upper phase), the main frame forward, rotating speed is 860rpm, then with 2.0mL/min, pumps into moving phase.Treat that moving phase flows out, when two-phase solvent reaches equilibrium state in post, get embodiment 2 gained Herba Polygoni Capitati secondary separation raw material thing 0.25g, be dissolved in isopyknic up and down mutually in, use syringe sampling, sample size 9.7mL.Divide and detected with the UV-detector convection current, the detection wavelength is 272nm, records color atlas simultaneously, the results are shown in Figure 2.Automatically collect flow point according to color atlas with fraction collector, every 3min collects a pipe.Disengaging time is more than 270min.
embodiment 6: purity check and Structural Identification
Utilize HPLC to analyze the composition of separating obtained each flow point of HSCCC in embodiment 5, judge its purity.Utilize UV, 1R, LC/MS, TOF-MS,
1h NMR reaches
13c NMR carries out structural analysis to products therefrom.
In the Fig. 2 separated at embodiment 5, collect the flow point of peak 1 and 2, respectively as flow point I and flow point II, utilize HPLC to analyze the purity of the separating obtained flow point I of HSCCC and flow point II.Flow point I is one matter by analysis, and purity is 96.2%; Flow point II is one matter, and purity is 98.4%, the results are shown in Figure 1.
In extra test, use the identical method of above embodiment 1～6 at some, unique difference is
in high speed adverse current chromatogram separates, use withthe two-phase solvent system of ethyl acetate-propyl carbinol-0.01mol/L dipotassium hydrogen phosphate solution three different ratios preparation.In a test, three's ratio is=3.36: 0.8: 5, component I and component I I reached 95% output when above respectively lower than 1.55mg with lower than 32.5mg in purity.In a test, three's ratio is=5.04: 0.8: 5, component I and component I I reached 95% output when above respectively lower than 1.25mg with lower than 27.5mg in purity.In a test, three's ratio is=4.2: 0.64: 5, component I and component I I reached 95% output when above respectively lower than 1.75mg with lower than 21.0mg in purity.In a test, three's ratio is=4.2: 0.96: 5, component I and component I I reached 95% output when above respectively lower than 1.20mg with lower than 25.8mg in purity.In a test, three's ratio is=3.78: 0.88: 5, component I and component I I reached 95% output when above in purity and are respectively 4.7mg and 72.5mg.In a test, three's ratio is=4.62: 0.72: 5, component I and component I I reached 95% output when above in purity and are respectively 4.4mg and 69.8mg.
embodiment 7: Structural Identification
Unknown material I and unknown material II to above acquisition carry out Structural Identification, and result is as follows:
unknown material I:
The yellowish brown powder, UV λ max (nm) MeOH:215.1,278.6,353.4.
IR(KBr)cm
-1：3432.56，2927.03，1677.55，1623.02，1516.28，1396.80，1305.54，1101.89。
TOF-MS：247.0230[M-H]
-，EI-MS：246.88[M-H]
-，218.86，190.95，172.95。
1H-NMR(400MHz，DMSO)δ(ppm)：7.73(1H，s，H-7)，3.18(2H，m，H-8)，2.50(2H，m，H-9)。
13C?NMR(101MHz，DMSO)δ(ppm)：195.49(C-10)，160.53(C-1)，149.47(C-5)，144.94(C-2)，144.18(C-4)，141.44(C-6)，140.23(C-3)，115.44(C-3a)，113.19(C-7a)，108.18(C-7)，32.97(C-9)，23.81(C-8)。
Above data are consistent with the brevifolin of document [6-8] report.
unknown material II:
Yellow powder, UV λ max (nm) MeOH:257.4,351.0.
IR(KBr)cm
-1：3445.77，3131.92，1650.76，1358.95，1263.86，1138.46，1067.13，994.37，951.73，863.89，766.24，618.59。
TOF-MS：447.0932[M-H]
-。
1H-NMR(400MHz，DMSO)δ12.66(1H，s，OH-5)，10.89(1H，s，OH-7)，9.72(1H，s，OH-4’)，9.34(1H，s，OH-3’)，7.30(1H，d，J＝2.1Hz，H-2’)，7.25(1H，dd，J＝2.1，2.2Hz，H-6’)，6.88(1H，d，J＝8.3Hz，H-5’)，6.39(1H，d，J＝1.9Hz，H-8)，6.21(1H，d，J＝1.9Hz，H-6)，5.25(1H，d，J＝1.4Hz，H-1”)，3.14～4.95(4H，m，H-2”，5”)，0.81(3H，d，J＝6.0Hz，H-6”)。
13C?NMR(101MHz，DMSO)δ(ppm)：177.7(C-4)，164.1(C-7)，161.2(C-5)，157.2(C-9)，156.4(C-2)，148.4(C-4’)，145.2(C-3’)，134.2(C-3)，121.1(C-6’)，120.8(C-1’)，115.6(C-5’)，115.4(C-2’)，104.0(C-10)，101.8(C-1”)，98.6(C-6)，93.6(C-8)，71.1(C-4”)，70.5(C-3”)，70.3(C-2”)，70.0(C-5”)，17.4(C-6”)。
The Quercitroside of above data and document [3,9] report is consistent.
In further studying, the inventor finds, using component I is that above purity reaches 96.2% brevifolin product in contrast, use HPLC condition mentioned above, the heat of 10 different batches that the inventor is obtained according to the method for making in the 992nd page of " heat is drenched clear particle " item of version Chinese Pharmacopoeia in 2010 is drenched clear particulate product and is detected, result shows that 10 batches of clear particulate product of heat pouring all detect brevifolin, and between the measured value of every batch and average, difference is no more than 20%.
The present invention is in above experiment, and the Golden-rule that the solvent-applied system is selected has been explored 12 separation systems, while finding to utilize ammoniacal liquor or strong acid weak base salt that the pH value of system is adjusted to 7.5 left and right, can obtain preferably α value.Find in actual tests, contain ammoniacal liquor and NaHCO
3system pH unstable, easily along with the variation of temperature, change, the appearance time of material to be separated is postponed greatly, affect separation and purification.Therefore select the ethyl acetate that pH is comparatively stable: propyl carbinol: 0.01mol/L dipotassium hydrogen phosphate solution system is separated.Investigated the different ratios of ethyl acetate, propyl carbinol and 0.01mol/L dipotassium hydrogen phosphate solution on the basis of this system, after having considered the factors such as resolution and disengaging time, final definite ethyl acetate that adopts: propyl carbinol: the 0.01mol/L dipotassium hydrogen phosphate solution=within 4.2: 0.8: 5, system is separated.And flow phase flow velocity, swivel pipe rotating speed to be optimized rear discovery flow rate of mobile phase be 2.0mL/min, separate best results when the swivel pipe rotating speed is 860rpm.Can from 0.25g Herba Polygoni Capitati secondary separation raw material thing, isolate 5.0mg brevifolin and 78.9mg Quercitroside under this separation condition.
Herba Polygoni Capitati is the very wide herbal medicine of a kind of application clinically, but also few about pertinent literature and the report of Herba Polygoni Capitati fundamental research.First Application high-speed countercurrent chromatography of the present invention extracts and has separated a new constituent monomer and another compound monomer from Herba Polygoni Capitati water extraction powder, through Structural Identification new discovery composition, is brevifolin; Another compound is Quercitroside.The systematic study that this research is the Herba Polygoni Capitati chemical composition provides a kind of efficient method, is suitable for the separation and Extraction of Herba Polygoni Capitati and Effective Component of Chinese Medicine, the preparation of effective constituent monomer, identification research.Definite and its pharmaceutical research of the preparation that this research is new standard material in Herba Polygoni Capitati, efficient part is laid a good foundation.
Following documents and materials are incorporated to this paper with its full content, and contributing to has more fully and understand the present invention:
[1] WuDetermined Ju, Wang Deren. the research of Herba Polygoni Capitati chemical composition [J]. herbal medicine, 1985,16 (4): 5.
[2] Liu Zhijun, relative is advanced, Zhu Danni, etc. Herba Polygoni Capitati chemical composition and anti-oxidant activity research [J]. Chinese medicinal materials, 2008,31 (7): 995.
[3] in bright, Li Zhanlin, Li Ning, etc. the chemical composition of Herba Polygoni Capitati [J]. Shenyang Pharmaceutical University's journal, 2008,25 (8): 633.
[4] Li Yongmei, Gong Yuan. the chemical composition of Herba Polygoni Capitati and pharmacological research progress [J]. Guizhou University's journal (natural science edition) .2007,24 (2): 205.
[5]Ito?Y，Golden?rules?and?pitfalls?in?selecting?optimum?conditions?for?high-speed?counter-current?chromatography[J].J?Chromatogr?A，2005，1065：145.
[6], Tian Ying, Sun Limin, Liu Xiqiao, etc. Chinese medicine Humifuse Euphorbia Herb phenolic constituent [J]. CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2010,35 (5): 613.
[7] Wang Xiaolan, Yu Kaibei, Peng Shulin, etc. the chemical constitution study of Herba Acalyphae over-ground part [J]. CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2008,33 (12): 1415.
[8] Lin Jia, Li Yan, Xu Lizhen. the chemical constitution study of Pomegranate Leaf [J]. Central-South pharmacy, 2005,3 (2): 70.
[9]Alessandra?B，Matteo?P，Rokia?S，et?al.Chemical?composition?and?antioxidant?activity?of?phenolic?compounds?from?wild?and?cultivated?Sclerocarya?birrea(Anacardiaceae)leaves[J].J.Agric.Food?Chem.2003，51：6689；6689。
Claims (10)
1. drench from heat the method for extracting brevifolin and/or Quercitroside the raw material Herba Polygoni Capitati aqueous extract of clear particle, the method comprises the following steps:
(1) get the Herba Polygoni Capitati aqueous extract, by 60～95% alcohol immersion 5～24 hours, supersound process 10～120min, filter the filtrate evaporation drying, the resistates water dissolution, filter, filtrate is extracted with ethyl acetate to upper strata colourless, and ethyl acetate layer is merged, evaporation drying, obtain ethyl acetate extract;
(2) step (1) gained ethyl acetate extract is loaded in high-speed counter-current chromatograph (HSCCC), use normal hexane: ethyl acetate: methyl alcohol: water is usingd the mixture of volume ratio 1:4.5～5.5:0.9～1.1:4.5～5.5 and is separated as the HSCCC solvent systems, collects the flow point of 40～90min; Repeat in case of necessity the operation of this separation and collection; Described flow point is steamed and desolventizes, obtain Herba Polygoni Capitati secondary separation raw material thing;
(3) in separating funnel, by ethyl acetate: the volume ratio of the dipotassium hydrogen phosphate solution of propyl carbinol: 0.01mol/L=3.78～4.62:0.72～0.88:5 configures the two-phase solvent system of about 1500mL, stratification becomes to be called the upper phase of stationary phase and is called the lower phase of moving phase, ultrasonic degas;
(4) adopt the type of elution of unicoil, head-to-tail, at first make pipeline be full of stationary phase, the main frame forward, speed is 800～900rpm, then pumps into moving phase, flow velocity is 1.5～2.5mL/min, treat that moving phase flows out, and when two-phase solvent reaches equilibrium state in post, gets step (2) gained Herba Polygoni Capitati secondary separation raw material thing, be dissolved in isopyknic up and down mutually in, use syringe sampling;
(5) divide and detected with the UV-detector convection current, the detection wavelength is 270～275nm, records color atlas simultaneously, according to color atlas, with fraction collector, automatically collects flow point, and every 3min collects a pipe; Disengaging time is more than 270min;
(6) collect between 30～40min go out peak flow point as flow point I, go out the flow point at peak between 200～260min as flow point II;
(7) remove respectively the solvent of flow point I and flow point II, flow point I and flow point II correspondingly obtain respectively component I and component I I, and optionally further refining to component I and component I I respectively, component I is brevifolin, and component I I is Quercitroside.
2. according to the process of claim 1 wherein that in step (1), ethanol used is 70～90% ethanol.
3. according to the process of claim 1 wherein that in step (1), the time of alcohol immersion is 8-15 hour.
4. according to the process of claim 1 wherein that in step (1), the time of supersound process is 15-60min.
5. according to the process of claim 1 wherein that in step (2), described HSCCC solvent systems is normal hexane: ethyl acetate: methyl alcohol: water is with the mixture of volume ratio 1:5:1:5.
6. according to the process of claim 1 wherein in step (2), collect the flow point of 45～80min.
7. according to the process of claim 1 wherein that in step (3), the ratio of described two-phase solvent system is 4.2:0.8:5.
8. according to the process of claim 1 wherein that in step (4), the rotating speed of main frame forward is 860rpm.
9. according to the process of claim 1 wherein that in step (4), the flow velocity that pumps into moving phase to pipeline is 2mL/min.
10. according to the process of claim 1 wherein that in step (5), described detection wavelength is 272nm.
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